PROJECT SUMMARY A critical challenge laid out in the NHLBI Strategic Vision is the development of single-cell analytics to assess health and resiliency. An area where single-cell methods are lacking is in the measurement of platelet reactivity. Specifically, the link between structural heterogeneity and function heterogeneity in platelets is poorly defined. This is a problem because platelets are regulators of bleeding, thrombotic, and inflammatory disorders, and we lack markers of resilience against these disorders. The long-term goal of this line of research is to develop technology that can reveal the mechanistic links between structural and functional heterogeneity in blood cells. The overall objective of this proposal, which is the first step along this continuum of research, is to develop an analytical approach to measure the relationships between mitochondria mass and RNA content and agonist-induced activation at the single platelet level and use that approach to measure changes from individuals from young to old age. Additionally, this approach should allow for pulsatile presentation of agonists because studies in other cells have shown pulsatile stimuli can synchronize response, better identifying subpopulations, and detect low and high frequency band pass filters in signal transduction. The rationale for this approach is that most clinical assays of platelet function measure ensembles of platelets that are unable to discriminate the role of different platelet subpopulations, nor are they able to tie structure and function at the single cell level. We will meet our overall objective with two specific aims: 1) Fabricate and test encapsulated platelets in cast (EPIC) hydrogels to quantify single cell structural and functional heterogeneity; and 2) Measure changes in platelet structure and function with age. We will develop a hydrogel-based platform to rapidly encapsulate fresh platelet isolates and measure the number of organelles or RNA content and correlate these measures with intracellular calcium dynamics and activation markers in response to pulsatile presentation of agonist(s). Using the EPIC platform, we will measure platelet structure and functional changes in platelet from childhood to old age in both sexes. We will test the hypothesis that mitochondrial mass increases with age and correlates with platelet hyperactivity. These studies will be compared to a comprehensive set of platelet phenotyping assays including platelet aggregometry, microfluidic flow assays, flow cytometry, and bioenergetics. The proposed research is innovative, in our opinion, because it represents a substantive departure from the status quo by developing a platform for studying thousands of single platelets in response to dynamic (temporally-modulated) stimuli in minutes. These contributions will be significant because they are expected to provide the means to study normal biological function in platelets in terms of their ability to sense, integrate, and respond to environmental stimuli.